Gut microbiota plasticity in insular lizards under reversed island syndrome

Animals living on small islands are more drastically exposed to environmental changes, such as food or water starvation, and rapid temperature shifts. Facing such conditions, and probably thank to adaptive plasticity mechanisms, some animals display a Reversed Island Syndrome (RIS), a suite of traits, including skin pigmentation, voracity, sexual dimorphism, showed differently from mainland relatives. Here, we analyse a so far poorly explored aspect of RIS: the effect of this on the microbiota composition of host Italian wall lizard (Podarcis siculus), strongly influenced by the animal’s lifestyle, and conditioning the same. We compare mainland and island populations, assessing the difference between their microbial communities and their response under unexpected food, experimentally provided. Our observations showed a significant difference in microbiota communities between island and mainland groups, depended mainly from changes in relative abundance of the shared genera (difference due to decrease/increase). Exposure to experimental diet regimes resulted into significative reshaping of bacterial composition of microbiota and a greater variation in body mass only in the island population. Our results could be an evidence that gut microbial community contributes to adaptive plasticity mechanisms of island lizards under RIS to efficiently respond to unexpected changes.

During experimental period, fed island lizards ate in average 19.54 ± 6.5 worms while fed mainland lizards consumed 16.00 ± 13.51 worms in average. Sequencing reads report. A total of 5,070,956 short raw reads (35- Table S1 online). The raw reads were separately processed and 2,016,960 filtered sequences (mean × sample ± SD: 57,627.43 ± 14,765.14) resulted after the bioinformatics analysis of the data, reaching 1,965,572 sequences (mean × sample ± SD: 56,159.20 ± 14,423.55) considering only the non-chimeric ones (Supplementary Table S1 online). The analysis of blank-negative water samples and mock communities controls did not reveal any inconsistencies in the expected profiles.
The Observed OTUs, Chao1, and Shannon rarefaction curves reached the plateau for all samples, showing that the estimates of species richness were stable and unbiased and the sequencing depth was sufficient for capturing a majority of microbial diversity and differences in microbial communities in the samples (Fig. S1a-c, Supplementary information online).
The two fasting populations differed significantly (one-way permutational multivariate analysis of variance, PERMANOVA test, p: 0.003) considering the bacterial communities at the genus taxonomic level. Indeed, Principal Coordinates Analysis (PCoA) accounted for 42.34% of variance, taking into account the first and the third axes (Fig. 2a). The ordination by this analysis produced two separate clusters, whose difference depended most of all from the contribution of Eisenbergiella (contribution of 10.64%), Enterobacter (9.07%), Bacteroides (8.02%) and Akkermansia (6.81%), with 60.07 of overall average dissimilarity (Fig. 2b and Supplementary Table S4 online). Furthermore, they shared 84 bacterial genera, with 8 and 25 exclusive for the gut microbiota of island lizards and mainland lizards, respectively (Fig. 2c and Supplementary Table S5 online).
The gut microbiota of IL lizards showed 92 bacterial genera, of which Eisenbergiella (13.86% ± 0.08) and Bacteroides (11.05% ± 0.07) were on average the most abundant ones, meanwhile for the MA gut microbiota, on 109 genera identified, Bacteroides (16.22% ± 0.08), and Enterobacter (10.04% ± 0.13) resulted to be on average the most abundant ones. All of the other genera showed an average occurrence lower than 10% (Supplementary  Table S6 online).
Comparing MA vs IL microbiota of fasting lizards by a post-hoc analysis, a total of 21 genera were found to be differentially represented, with 6 genera only present in the gut microbiota of mainland animals and 2 genera exclusively present in the gut microbiota of island lizards. These exclusive genera included Gram-negatives (Prevotella, Zooglea, Rivicola, Sulfuricum, Dysgonomonadaceae) and Gram-positive (Streptococcus, Eubacterium eligens, Pelosinus) bacteria (Table 1).
The 13 shared genera differentially represented in the two groups were Faecalibacterium, Lactobacillus, Pseudomonas, Ruminococcus 1, Lachnospira and Cutibacterium, more abundant in the gut microbiota of MA lizards, and Intestinimonas, Angelakisella, Hydrogenoanaerobacterium, Butyricicoccus, Oscillospira, Clostridium sensu stricto 19 and Eisenbergiella, more represented in IL animals (Fig. 2d). The Pearson's chi-square test, performed considering all 21 genera and permutation with 9999 replicates, indicated that observed differences between IL and MA groups were independent from random variable (Chi-square value: 11.415, degrees of freedom: 4, p value: 0.002). Furthermore, this was confirmed by Fisher´s exact test (p value: 0.003).

Variation of gut microbiota communities' composition under unexpected food change.
To characterize the plasticity of island and mainland lizards, we imposed an unusual diet, and then compared the After the same experimental treatment, both microbiota of fed island lizards (IL_F) and fed mainland lizards (MA_F) rearranged in a different way compared with the original corresponding microbiota community (fasting IL and MA, respectively), however statically significative difference was revealed only for insular microbiota (Fig. 3). In particular, PCoA generated using the bacterial genera of the four groups accounted for 36.80% of variance (Fig. 3), with microbiota of island lizards that split significantly from microbiota of fed animals (one-way PERMANOVA, p < 0.001; see Supplementary Table S7 online for details), whereas MA_F was almost completely overlapping MA in the plot (one-way PERMANOVA, p: 0.27).
The Similarity Percentages Species Contributions (SIMPER) analysis considering IL and IL_F populations ( Fig. 4a) showed an overall average dissimilarity of 59.93, with Eisenbergiella (contribution of 9.36%), followed by Akkermansia (6.58%), Bacteroides (6.12%) and Romboutsia (5.04%), that were the most involved genera in the differentiation between microbiota of the island groups (Supplementary Table S8 online). Meanwhile, Enterobacter (11.4%) was the genus mainly involved in determining the dissimilarity between MA and MA_F groups ( Fig. 4b and Supplementary Table S9 online).
Even at the genus level, the variation showed in the island system concerned both the type, the number and the relative abundance of bacterial genera (Supplementary Table S6 online). Indeed, of 115 total bacterial genera, microbiota of IL and IL_F lizards shared 91 genera, and only one (Candidatus Stoquefichus) was exclusive of fasting lizards, while 23 were unique of fed island samples ( Fig. 4c and Supplementary Table S10 online). This trend was also found in the mainland lizard groups even if less evidently. Indeed, of 116 genera, 106 were shared between the microbiota of MA and MA_F lizards, 3 genera were exclusive of microbiota of MA lizards and 7 were exclusive of microbiota of MA_F group ( Fig. 4d and Supplementary Table S11 online).
The 3 genera differentially represented in the microbiota of MA vs MA_F groups (Acinetobacter, Sphingobacterium and Staphylococcus) were all more abundant in MA_F than in MA (Fig. 5b). Interestingly, members   www.nature.com/scientificreports/ of the Sphingobacterium and Staphylococcus increased both in IL_F and MA_F groups suggesting a direct link between their abundance and the specific diet. Analysis of the α-diversity descriptors, calculated at genus taxonomic level, indicated that the unusual diet caused an increased microbial diversity both for the microbiota of IL_F and MA_F lizards (Fig. 6), although statistically significant differences were observed only for Richness (Kruskal-Wallis, p: 0.007) between IL and IL_F groups (H: 8.597, p: 0.003) and IL and MA_F (H: 10.599, p: 0.001).  www.nature.com/scientificreports/ Weight adjustment. During experimental period, fed island lizards and mainland lizards ate a comparable number of moth larvae (mean ± SD: 19.54 ± 6.5 vs 16.00 ± 13.51; not significant difference by ANOVA test, p: 0.47). It is interesting to note that this food surplus provided to both populations generates a greater variation in body mass only for island lizards, probably as a result of a reorganization of their microbiota based on this unexpected food source (Fig. 7). Only a single fed island sample (IL_35_F) showed a reduction in weight after the experimental treatment (delta body mass: − 4 g). Thus, we discarded it from this analysis by attributing this distortion to an error in weight measurement. Nevertheless this, the correlation between weight variation and ingested moths was always higher for fed island lizards (R 2 0.707; t-score: 4.11; p: 0.004) than for mainland lizards (R 2 = 0.5644; t-score: 2.788; p: 0.03) (Fig. 7).

Discussion
Our experimental model turns out to be optimal for revealing how RIS affects the possible changes of the gut microbiota community of lizards, as testified to the significative differences between microbiota composition of IL and MA. Variation in microbiota of lizards under experimental condition may depend on diet (i.e. omnivorous vs herbivorous) and host species 2 . Our populations belong to the same omnivorous species and were collected during the same season, thus, we could assume that factors as (i) the introduction of "food" as a sudden and unexpected change, (ii) the simplification of the diet in terms of number of ingested items and, (iii) the fasting, affect the variation in microbiota of our experimental groups. It is interesting, and worthy of further study, that these constraints introduce an increase in microbiota diversity.
Gut microbial composition results from a mix of commensal "core" genera (sensu 39,40 ) and a flexible pool of beneficial microbes which may confer a selective advantage during periods of stress or rapid adaptation 41 . The analysis of the latter component can be an indicator of the adaptive potential of a species or a population. It allows to speculate on plasticity and ecological segregation, particularly for species that are unable to make large displacements and subjected to the ecological conditions where they are confined.
Our findings showed that the microbiota diversity depends mainly on different relative abundances of the shared genera (difference due to decrease/increase) rather than changes in bacterial genera composition (difference due to substitution). For example, the gut microbiota of mainland and island lizards, although characterized by a different number of bacterial genera (92 vs 109, respectively), shared a core of 84 genera, showing similar values of alfa-diversity index. All this was affected probably by the dominance of few bacteria (Bacteroides and Enterobacter for MA lizards and Eisenbergiella and Bacteroides for IL lizards) characterized by the highest average relative abundance, followed by all the other genera showing an average relative abundance less than 10%.
The gut microbiomes of squamate reptiles have been largely overlooked in terms of ecology 42 when compared to the other vertebrates 43 . Indeed, less than 10% of studies investigating the gut microbiota communities of vertebrates were conducted on non-mammalian hosts 42 and some of these indicate that squamate reptile microbiota would be more similar to those of fishes and birds than those of mammals 43 .
In our study, the more abundant genera in IL lizards belong to four Families: Oscillospiraceae (Hydrogenoanaerobacterium, Oscillospira), Ruminococcaceae (Intestinimonas, Angelakisella), Clostridiaceae (Butyricicoccus, Clostridium sensu stricto 19) and Lachnospiraceae (Eisenbergiella). In particular, Oscillospira is commonly found in the intestinal microbiota of vertebrates with a potential role in the digestion of cellulose and in the fermentation of fibres in herbivorous, including reptiles 42,[44][45][46] . All members of the Oscillospira genus have been shown to use animal-derived glycans (i.e. glucuronate) to produce butyrate 47 . The abundance of Oscillospira increased in response to prolonged fasting in lizards and other vertebrates as well 48 , probably promoting the degradation of some glycans (i.e. fucose, sialic acids, and glucuronic acid) of the host 48 . Accordingly, in our study, we observed an increase of Oscillospira in fasting island lizards compared to fed island lizards (delta-variation: 0.006). This probably represents a response to the unpredictable conditions on the island that lead these lizards to cope with periods of prolonged fasting.
MA populations showed lower level of Oscillospira than IL, and this decreased after food providing but with slight variation if compared to IL microbiota. Arguably, the differential level of Oscillospira and variations in a shorter time interval than mainland conspecifics are in agreement with RIS 23 .
Members of the Intestinimonas and Angelakisella are also not known in detail, however they are considered specialized in the digestion of cellulose with an essential role in the fermentation of fibres in herbivores, including reptiles 2,44 . This could be related to a greater herbivorous behaviour in island lizards to survive during periods of low animal prey availability.
The genus Clostridium sensu stricto, anaerobic Gram-positive spore formers commonly found in human and animal guts 49 , and Eisenbergiella, isolated from human blood and faecal samples 50 , were found involved in the maintenance of gut homeostasis and in modulating the functional activities of the cells of the immunological system 49 . In particular, the latter was strongly correlated with increased levels of TNFα and IFNα in chickens' intestinal epithelial cells 51,52 . All of this is in line with the RIS that predicted a more active immune system in island lizard than in mainland ones 19 .
Bao and co-workers reported an increase in Eisenbergiella after infection by Echinococcus granulosus in mice 53 . Furthermore, Dipineto and collaborators, analysing parasites in lizard populations on mainland and island from the same as our study area, showed the presence of Coccidae only in faeces of island lizards 54 . Interestingly, our results indicated that the relative abundance of Eisenbergiella is significantly greater in the microbiota of IL than MA.
Among the bacterial genera more significant discriminative of MA lizards microbiota we found Enterobacter, ubiquitous 55 and also part of the commensal microflora of animals 56 and of the human gut 57 www.nature.com/scientificreports/ possible explanation for this could be that mainland lizards have more opportunities of contacting with humans or animal (wild and domestic) waste rather than the island lizards. Indeed, the latter lives on Licosa islet, only occasionally frequented by birds or humans to swim or boat during the summer. This hypothesis is according to the isolation condition that affect lizards under RIS and opens up interesting hypotheses on the synanthropic 59 commensalism of this animal. In our study, we did not perform a direct characterisation of microbial community of moth. However, to select which bacteria could be introduced by the food supplied, we have extracted the items shared uniquely by the fed lizards and absent in island and mainland fasting ones, that were Candidatus and Rickettsiella.
The former characterises the microbial community of vertebrates (i.e. 60 ), and therefore we excluded that its presence in gut microbiota of lizards was due to a conditioning by the microbiota of the moth.
Rickettsiella was found in a great variety of arthropod species [61][62][63] , including also some parasites adherent to tissues of Podarcis sp. 64 . Therefore we cannot define with certainty whether this bacterial genus is part of the microbiota of lizards, whether it derives from the infection of these by parasites or from the diet provided in captivity. Nevertheless, Rickettsiella genus was extremely underrepresented in the microbiota community of fed mainland (mean ± SD: 0.0000053 ± 0.000102) and fed island lizards (mean ± SD: 0.01 ± 0.030). Further investigations into the ecology of allochthonous microbes would provide more insight into the assembly of the gut microbiota of lizards.
The supply of unexpected food changed, qualitatively and quantitatively, the microbiota composition both of mainland and island populations; however, 91 and 106 bacterial genera were still shared between fasting and fed lizards, in insular and mainland intra-comparation, respectively (Fig. 4c,d). This result further suggests that the lizards maintained their core gut microbial communities during the 5 days of experiment, as demonstrated also by Kohl et al. 2 that showed how their captive lizards, treated for 8 weeks, retained ~ 65% of their wild bacterial microbiota. Probably, the consistency of this core will reduce over time, and the faster variation that we detected could be explained by the syndrome that island lizards undergo.
Interestingly, comparing the microbiota composition of fasted and fed lizards, we revealed statistically significant differences only for island populations. This variation could be more evident for island system because of the lower richness in bacterial genera of the microbiota compared to that of mainland lizards (15% less). However, it should also be considered that these two groups shared 77% of the microbial bacterial groups. These results could be an evidence of the ability of the island lizards (under RIS) to respond more significantly than mainland populations to drastic and unexpected changes, by adopting adaptive plasticity mechanisms 11,21 , adding another important element to the theoretical basis of the syndrome 18,19,22,23 .
Our contribution opens the doors to future investigations aiming at shedding light on a mechanism as interesting as the plasticity of populations in highly unpredictable environmental conditions, such as those on small islets, revealing how gut microbial communities may be impacting the ecology and evolution of island lizard hosts.
Probably, the attitude of island lizards to have a high feed intake (according to the RIS) and to use a variety of food items, such as the regurgitation of gulls (personal observation), could explain plasticity observed in the microbiota of island individuals.
Furthermore, the island lizards showed to be bolder than mainland relatives (according to the RIS) and, thanks to this, they may have the opportunity to explore more complex ecological niches. This circumstance could promote a modelling of the microbiota in relation to the resources encountered. These etho-ecological peculiarities of the island lizards could be linked to their microbiota, which would be better adapted to exploit the unexpected resources, as suggested by the weight increase recorded in the experimental group (IL_F).
Moreover, there are probably key genes involved in the plasticity and peculiarity of the microbiota of insular lizards. We previously described the transcriptome of P. siculus 22 considering mainland and island specimens, and this could represent an opportunity to deep the information about differential expressed genes underling host-microbiome interactions.

Material and methods
Study area. Our survey was conducted on the islet of Licosa and on the facing mainland, during the summer. The islet of Licosa (40°15′N. 14°54′E. Salerno, South Italy) is 400 m away from the closest mainland (Punta Licosa, 18. 40°50′N. 14°15′E) (Fig. 1) and is dominated by Pistacia lentiscus with naked and stony shores. On the island there are small populations of rats (Rattus sp.), Mediterranean house geckos (Hemidactylus turcicus) and very few nesting passerine birds. However, the island predators of lizards are potentially represented by gulls (Larus michahellis and Chroicocephalus ridibundus), very rarely kestrels (Falco tinnunculus), and carrion crows (Corvus corone).
The mainland is characterised by luxuriant Mediterranean scrub with olive tree cultivations, rural buildings and stone walls. Mainland lizards are preyed on by mostly birds as tawny owls (Strix aluco), little owls (Athene noctua), kestrels, red-backed shrikes (Lanius collurio) and carrion crows as well as by terrestrial predators like rats, grass snakes (Natrix natrix), green whip snakes (Hierophis viridiflavus), and feral cats (Felis catus).
The food items of island and mainland populations of lizards were almost similar, consisting mainly of arthropods, such as Diptera, Isopoda and Coleoptera, larvae of Lepidoptera ( 31,65-67 and our field observations) and occasionally of small vertebrates 68,69 .
Moreover, our field observations showed that island lizards supplement their diet with gull regurgitates, chicks of passerines, or they can exploit occasional phenomena such as the migrations of butterfly that stopover on the island. The captured lizards were transported to the research station and individually placed in sterile terrariums with autoclaved soil, and kept at 23-27 °C with natural day/night periods. Lizard boxes, accessorized with habitat decorations including wood branches and rocks, were separated with cardboards so the lizards could not see one another, to avoid potential stress due to interaction. Each experimental group of lizards was placed in a separate room.
All lizards used for the analyses were > 1 year old, aged according to the snout-vent length (SVL), which is correlated with age 18,70 . Island lizards had a SVL of 7.5 ± 0.4 cm while SVL for mainland lizards was 7.8 ± 0.3. The lizards were weighed to the nearest 0.01 g on Mettler precision balance (Mettler Toledo) before and after the experimental trials.
The samples were divided into four experimental groups: two lizards groups held without supplied food (fasting island lizards: IL = 9 and fasting mainland lizards: MA = 10) and two lizards groups fed and watered ad libitum for 5 days (fed island lizards: IL_F = 13 and fed mainland lizards: MA_F = 11). Zero time (T 0 ) corresponded to the first day of capture of a lizard and, since not all the lizards were caught on the same day, T 0 does not necessarily synchronized among the specimens.
The water was sterilized under UV lamp for 1 h before the using 71 . The food was represented by Honeycomb moth (Galleria mellonella) caterpillars, not present where lizards lived, bought in a company specializing in laboratory animals. The moth was selected as an exotic diet to evaluate the ability of lizards to adapt to a new dietary regimen.
We used faecal samples as a proxy for gut microbiota considering that they provide a complete view of hindgut bacterial communities in lizards useful for microbial inventories 2,72,73 . In particular, during the experimental period, we monitored the animals at least every 5 h so that we sampled only fresh faecal material (< 5 h). For fed animals, the faecal material deposited in the terrarium soon after catching were discarded. The scats were collected using sterilized equipment and stored immediately in 2 ml microcentrifuge tubes frozen at − 20 °C. Each lizard laid on average 4.13 ± 1.43 excrements in 5 days (in particular, IL = 3.61 ± 1.11; IL_F: 5.13 ± 1.8; MA = 3.61 ± 0.92; MA_F = 3.93 ± 1.14). The faeces of a single individual during the 5 experimental days were pooled every time in the same tube. At the end of experimental period, the pools were transported in containers, at controlled temperature, to the laboratory. Here, they were stored at − 20 °C until DNA extraction and sequencing, performed at most three day after arrival.
Lizards were collected with the authorization of the Ministry of the Environment and Protection of Land and Sea, and experimental procedures, approved also by the institutional review board "Societas Herpetologica Italica", were performed according to Italian law. This study follows the recommendations reported in the ARRIVE guidelines 74,75 and all the methods were performed in accordance with the relevant guidelines and regulations. At the end of 5 experimental days for all lizards, they were released at the point of capture. Microbiota identification by 16S rRNA gene amplification, sequencing, and data analysis. A fragment of about 190 bp of 16S rRNA gene V3 region was amplified using Probio_Uni (5′-CCT ACG GGRSGCA GCA G-3′) and Probio_Rev (5′-ATT ACC GCG GCT GCT-3′) primers, and then sequenced on Illumina MiSeq platform at GenProbio srl (www. genpr obio. com), according to the protocol described in 76 . The sequencing included blank-negative water samples and specific mock communities (ZymoBIOMICS HMW DNA Standard) as additional quality check control.
After demultiplexing, the reads of each sample were trimmed and filtered to remove low quality and chimeras, and processed using a script based on the QIIME software suite 77 . Paired-end reads were assembled to reconstruct the complete Probio_Uni /Probio_Rev amplicons. The sequences between 140 and 400 bp in length and mean sequence quality score > 20 were retained, removing mismatched primers and sequences with homopolymers > 7 bp. 16S rRNA Operational Taxonomic Units (OTUs) were defined at ≥ 99% sequence homology using uclust 78 and OTUs with less than 10 sequences were filtered. All reads were classified to the lowest possible taxonomic rank using QIIME 77 and the SILVA database v. 132 clustered at 99% identity as reference dataset 79 .
The results are expressed as percentual frequency for each sample, defined by the ratio between the number of reads in each OTUs and the total number of reads. Representative OTUs of the same genus were added together to have the taxonomic profile at the genus level. Extremely poorly represented taxa (relative abundance < 0.002%) were discarded from the subsequent elaborations, according to 80,81 . Finally, we calculated the number of observed OTUs, Chao1 and Shannon indices.
Statistical analysis. Community membership and structure were represented by a PCoA using Bray-Curtis dissimilarity 82 . One-way PERMANOVA was performed to test significance of multivariate analyses.
We assessed which taxa are primarily responsible for an observed difference between groups of samples 83 using SIMPER with Bray-Curtis dissimilarity, in Past v. 3.2. software 84 .
Sample α-diversity was calculated using Richness 85  www.nature.com/scientificreports/ significant differences between the groups, we performed a Kruskal-Wallis test, followed by pairwise Wilcoxon tests using the software R 88 .

Data availability
All 16S rRNA gene sequences produced for this study are available in the Sequence Read Archive (SRA) under accession number PRJNA791286.